Metal Binding Affinities of Arabidopsis Zinc and Copper Transporters: Selectivities Match the Relative, but Not the Absolute, Affinities of their Amino-Terminal Domains,

Abstract

HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P(1B)-type ATPases in the simple plant Arabidopsis thaliana. The first two transport Zn(2+), and the third transports Cu(+). Each protein contains soluble N-terminal metal-binding domains (MBDs) that are essential for metal transport. While the MBD of HMA7 features a CxxC sequence motif characteristic of Cu(I) binding sites, those of HMA2 and HMA4 contain a CCxxE motif, unique for plant Zn(2+)-ATPases. The three MBDs HMA2n (residues 1-79), HMA4n (residues 1-96), and HMA7n (residues 56-127) and an HMA7/4n chimera were expressed in Escherichia coli. The chimera features the ICCTSE motif from HMA4n inserted in place of the native MTCAAC motif of HMA7n. Binding affinities for Zn(II) and Cu(I) of each MBD were determined by ligand competition with a number of chromophoric probes. The challenges of using these probes reliably were evaluated, and the relative affinities of the MBDs were verified by independent cross-checks. The affinities of HMA2n and HMA4n for Zn(II) are higher than that of HMA7n by a factor of 20-30, but the relative affinities for Cu(I) are inverted by a factor of 30-50. These relativities are consistent with their respective roles in metal selection and transportation. Chimera HMA7/4n binds Cu(I) with an affinity between those of HMA4n and HMA7n but binds Zn(II) more weakly than either parent protein does. The four MBDs bind Cu(I) more strongly than Zn(II) by factors of >10(6). It is apparent that the individual MBDs are not able to overcome the large thermodynamic preference for Cu(+) over Zn(2+). This information highlights the potential toxicity of Cu(+) in vivo and why copper sensor proteins are approximately 6 orders of magnitude more sensitive than zinc sensor proteins. Metal speciation must be controlled by multiple factors, including thermodynamics (affinity), kinetics (including protein-protein interactions), and compartmentalization. The structure of Zn(II)-bound HMA4n defined by NMR confirmed the predicted ferredoxin betaalphabetabetaalphabeta fold. A single Zn atom was modeled onto a metal-binding site with protein ligands comprising the two thiolates and the carboxylate of the CCxxE motif. The observed (113)Cd chemical shift in [(113)Cd]HMA4n was consistent with a Cd(II)S(2)OX (X = O or N) coordination sphere. The Zn(II) form of the Cu(I) transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(Zn(II)-HMA7n)(m)]. Each Zn(II) ion occupied a distorted tetrahedral site formed from two Cys ligands of the CxxC motif of one HMA7n molecule and the amino N and carbonyl O atoms of the N-terminal methionine of another.

Metal Selectivity of the Escherichia coli Nickel Metallochaperone, SlyD

“…The Cu(I) salts were recrystallized as described previously to remove any trace amounts of Cu(II) and the resulting powder was dissolved in an Ar(g) saturated 30% (v/v) acetonitrile/water solution (17). The concentrations of the copper solutions were established by using bathocuproine sulfonate (Bcs) as described previously (18). Buffers for all metal assays were prepared with Milli-Q water that was then treated with Chelex-100 (Bio-Rad) and stirred in an anaerobic glovebox (O 2 < 1 ppm) for at least 24 h prior to use.…”

“…Titrations of 400 μM PAR with Zn(II) under identical conditions were used to calculate the amount of [Zn-PAR 2 ] formed in the competition described above. As the pH of the solution greatly affects the formation constant as well as the molar absorption coefficient (ε) of the Zn-PAR 2 complex, the metal-binding affinity of PAR under these particular buffer conditions was calibrated by using EGTA as described in detail by Zimmerman et al (18). Further details of the competition reactions are in the supplementary information (SI).…”

Influence of Soil Chemistry and Plant Physiology in the Phytoremediation of Cu, Mn, and Zn

“…It should be emphasized that the translocation of trace metals is a complex process involving multiple networks between membrane transporters and metals. Specialized proteins can transport elements with similar characteristics (e.g., oxidative state), inducing competition that results in deficiency, toxicity and/or accumulation in the above-ground plant tissues (Barberon et al, 2011;Zimmermann et al, 2009).…”

“…The P-type ATPases, known as heavy metal P-type ATPases (HMAs) in plants, include at least eight (HMA1-HMA8) identified members in Arabidopsis thaliana. AtHMA1 to AtHMA4 belong to the group implicated in divalent cation transport; AtHMA5 to AtHMA8 act on monovalent Cu ion transport (Zimmermann et al, 2009;Zorrig et al, 2011). Efflux of Cu into the vascular tissues is thought to occur through an HMA family transporter.…”

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

“…The Cys residues are responsible for metal coordination and can be involved in binding both monovalent (Ag ϩ and Cu ϩ ) and divalent metal cations (Cu 2ϩ , Cd 2ϩ , and Zn 2ϩ ) (16 -21). In plant Zn 2ϩ -ATPases the Cys-X-X-Cys conserved sequence is replaced by a Cys-Cys-X-X-Glu motif (9,12,22,23). Truncation of P1B-ATPase N-terminal metal binding domains or mutation in their metal binding sequences lead to reduced enzyme activity but does not affect the affinity of metal binding to transmembrane transport sites (24 -29).…”